Protein purification

ABSTRACT

A method for purifying a polypeptide by ion exchange chromatography is described which involves changing the conductivity and/or pH of buffers in order to resolve a polypeptide of interest from one or more contaminants.

This is a divisional of co-pending application No. 09/304,465 filed May3, 1999 which claims priority under 35 USC §119 to provisionalapplication No. 60/084,459 filed May 6, 1998, both disclosures of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to protein purification. In particular,the invention relates to a method for purifying a polypeptide (e.g. anantibody) from a composition comprising the polypeptide and at least onecontaminant using the method of ion exchange chromatography.

2. Description of Related Art

The large-scale, economic purification of proteins is increasingly animportant problem for the biotechnology industry. Generally, proteinsare produced by cell culture, using either mammalian or bacterial celllines engineered to produce the protein of interest by insertion of arecombinant plasmid containing the gene for that protein. Since the celllines used are living organisms, they must be fed with a complex growthmedium, containing sugars, amino acids, and growth factors, usuallysupplied from preparations of animal serum. Separation of the desiredprotein from the mixture of compounds fed to the cells and from theby-products of the cells themselves to a purity sufficient for use as ahuman therapeutic poses a formidable challenge.

Procedures for purification of proteins from cell debris initiallydepend on the site of expression of the protein. Some proteins can becaused to be secreted directly from the cell into the surrounding growthmedia; others are made intracellularly. For the latter proteins, thefirst step of a purification process involves lysis of the cell, whichcan be done by a variety of methods, including mechanical shear, osmoticshock, or enzymatic treatments. Such disruption releases the entirecontents of the cell into the homogenate, and in addition producessubcellular fragments that are difficult to remove due to their smallsize. These are generally removed by differential centrifugation or byfiltration. The same problem arises, although on a smaller scale, withdirectly secreted proteins due to the natural death of cells and releaseof intracellular host cell proteins in the course of the proteinproduction run.

Once a clarified solution containing the protein of interest has beenobtained, its separation from the other proteins produced by the cell isusually attempted using a combination of different chromatographytechniques. These techniques separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down along column, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different solvents. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through”.

Ion exchange chromatography is a chromatographic technique that iscommonly used for the purification of proteins. In ion exchangechromatography, charged patches on the surface of the solute areattracted by opposite charges attached to a chromatography matrix,provided the ionic strength of the surrounding buffer is low. Elution isgenerally achieved by increasing the ionic strength (i.e. conductivity)of the buffer to compete with the solute for the charged sites of theion exchange matrix. Changing the pH and thereby altering the charge ofthe solute is another way to achieve elution of the solute. The changein conductivity or pH may be gradual (gradient elution) or stepwise(step elution). In the past, these changes have been progressive; i.e.,the pH or conductivity is increased or decreased in a single direction.

SUMMARY OF THE INVENTION

The present invention provides an ion exchange chromatographic methodwherein a polypeptide of interest is bound to the ion exchange materialat an initial conductivity or pH and then the ion exchange material iswashed with an intermediate buffer at a different conductivity or pH, orboth. At a specific point following this intermediate wash, and contraryto ion exchange chromatography standard practice, the ion exchangematerial is washed with a wash buffer where the change in conductivityor pH, or both, from the intermediate buffer to the wash buffer is in anopposite direction to the change in conductivity or pH, or both,achieved in the previous steps. Only after washing with the wash buffer,is the ion exchange material prepared for the polypeptide molecule ofinterest to be eluted by the application of the elution buffer having aconductivity or pH, or both, which differ from the conductivity or pH,or both, of the buffers used in previous steps.

This novel approach to ion exchange chromatography is particularlyuseful in situations where a product molecule must be separated from avery closely related contaminant molecule at full manufacturing scale,where both purity and high recovery of polypeptide product are desired.

Accordingly, the invention provides a method for purifying a polypeptidefrom a composition comprising the polypeptide and a contaminant, whichmethod comprises the following steps performed sequentially:

(a) binding the polypeptide to an ion exchange material using a loadingbuffer, wherein the loading buffer is at a first conductivity and pH;

(b) washing the ion exchange material with an intermediate buffer at asecond conductivity and/or pH so as to elute the contaminant from theion exchange material;

(c) washing the ion exchange material with a wash buffer which is at athird conductivity and/or pH, wherein the change in conductivity and/orpH from the intermediate buffer to the wash buffer is in an oppositedirection to the change in conductivity and/or pH from the loadingbuffer to the intermediate buffer; and

(d) washing the ion exchange material with an elution buffer at a fourthconductivity and/or pH so as to elute the polypeptide from the ionexchange material. The first conductivity and/or pH may be the same asthe third conductivity and/or pH.

Where the ion exchange material comprises a cation exchange resin, theconductivity and/or pH of the intermediate buffer is/are preferablygreater than the conductivity and/or pH of the loading buffer; theconductivity- and/or pH of the wash buffer is/are preferably less thanthe conductivity and/or pH of the intermediate buffer; and theconductivity and/or pH of the elution buffer is/are preferably greaterthan the conductivity and/or pH of the intermediate buffer. Preferably,the conductivity and/or pH of the wash buffer is/are about the same asthe conductivity and/or pH of the loading buffer.

Preferably elution of the contaminant and of the polypeptide is achievedby modifying the conductivity of the intermediate buffer and of theelution buffer, respectively, while keeping the pH of these buffersapproximately the same.

The invention also provides a method for purifying a polypeptide from acomposition comprising the polypeptide and a contaminant, which methodcomprises the following steps performed sequentially:

(a) binding the polypeptide to a cation exchange material using aloading buffer, wherein the loading buffer is at a first conductivityand pH;

(b) washing the cation exchange material with an intermediate buffer ata second conductivity and/or pH which is greater than that of theloading buffer so as to elute the contaminant from the ion exchangematerial;

(c) washing the cation exchange material with a wash buffer which is ata third conductivity and/or pH which is less than that of theintermediate buffer; and

(d) washing the cation exchange material with an elution buffer at afourth conductivity and/or pH which is greater than that of theintermediate buffer so as to elute the polypeptide from the ion exchangematerial.

In addition, the invention provides a method for purifying an antibodyfrom a composition comprising the antibody and a contaminant, whichmethod comprises loading the composition onto a cation exchange resin,wherein the amount of antibody loaded onto the cation exchange resin isfrom about 20 mg to about 35 mg of the antibody per mL of cationexchange resin and, optionally, further comprising eluting the antibodyfrom the cation exchange resin. The method preferably further comprisesan intermediate wash step for eluting one or more contaminants from theion exchange resin. This intermediate wash step usually precedes thestep of eluting the antibody.

The invention further provides a composition comprising a mixture ofanti-HER2 antibody and one or more acidic variants thereof, wherein theamount of the acidic variant(s) in the composition is less than about25% and preferably less than about 20%, e.g. in the range from about 1%to about 18%. Optionally, the composition further comprises apharmaceutically acceptable carrier

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram showing how one could perform cation exchangechromatography by altering conductivity (e.g. to the NaCl concentrationsof Example 1 below) or by altering pH (e.g. to the pH values as shown inthe flow diagram).

FIG. 2 is a flow diagram showing how one could perform anion exchangechromatography by altering conductivity (e.g. to the Nacl concentrationsas depicted in the figure) or by altering pH (e.g. to the pH values asshown).

FIG. 3 is an absorbance trace from a cation exchange chromatography runof Example 1 at full manufacturing scale. Points at which the column iswashed with the different buffers described herein are marked witharrows.

FIG. 4 depicts recombinant humanized anti-HER2 monoclonal antibody(rhuMAb HER2) recovered in each chromatography fraction (calculated asthe percentage of the sum total of all fractions of the relevantchromatography). Flow through, wash steps, and prepool fractions are alleffluent samples collected from the onset of load to the initiation ofpooling. The pool fraction is the five column volume effluent sample ofelution starting at the leading shoulder's inflection point. Theregeneration fraction contains effluent captured from the end of poolingto the end of regeneration.

FIG. 5 shows the quality of rhuMAb HER2 in each cation exchangechromatography pool sample as evaluated by carboxy sulfon cationexchange high pressure liquid chromatography (CSx HPIEX). Peaks a, b,and 1 are deamidated forms of rhuMAb HER2. Peak 3 is nondeamidatedrhuMAb HER2. Peak 4 is a combination of C-terminal Lysine containing andiso-aspartate variants of rhuMAb HER2.

FIG. 6 shows the absorbance (280 nm) profiles of the 0.025 M MES/0.070 MNaCl, pH 5.6 wash for each chromatography. The mass of rhuMAb HER2applied to the cation exchange resin effects the peak's absorbance levelat the apex as well as the amount of buffer required to reach the apex.Due to minor peaks which occur (as best seen in the 30 mg/mL load) inthis wash, the apex is defined as absorbance levels of at least 0.5absorbance units (AU).

FIGS. 7A and 7B show the amino acid sequences of humMAb4D5-8 light chain(SEQ ID NO:1) and humMAb4D5-8 heavy chain (SEQ ID NO:2), respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions:

The “composition” to be purified herein comprises the polypeptide ofinterest and one or more contaminants. The composition may be “partiallypurified” (i.e. having been subjected to one or more purification steps,such as Protein A Chromatography as in Example 1 below) or may beobtained directly from a host cell or organism producing the polypeptide(e.g. the composition may comprise harvested cell culture fluid).

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. Preferably, the polypeptide is amammalian protein, examples of which include renin; a growth hormone,including human growth hormone and bovine growth hormone; growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; Protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 andCD20; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments and/or variants of any of the above-listedpolypeptides. Most preferred is a full length antibody that binds humanHER2.

A “contaminant” is a material that is different from the desiredpolypeptide product. The contaminant may be a variant of the desiredpolypeptide (e.g. a deamidated variant or an amino-aspartate variant ofthe desired polypeptide) or another polypeptide, nucleic acid, endotoxinetc.

A “variant” or “amino acid sequence variant” of a starting polypeptideis a polypeptide that comprises an amino acid sequence different fromthat of the starting polypeptide. Generally, a variant will possess atleast 80% sequence identity, preferably at least 90% sequence identity,more preferably at least 95% sequence identity, and most preferably atleast 98% sequence identity with the native polypeptide. Percentagesequence identity is determined, for example, by the Fitch et al., Proc.Natl. Acad. Sci. USA 80:1382-1386 (1983), version of the algorithmdescribed by Needleman et al., J. Mol. Biol. 48:443-453 (1970), afteraligning the sequences to provide for maximum homology. Amino acidsequence variants of a polypeptide may be prepared by introducingappropriate nucleotide changes into DNA encoding the polypeptide, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequence of the polypeptide of interest. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the polypeptide, such as changing thenumber or position of glycosylation sites. Methods for generating aminoacid sequence variants of polypeptides are described in U.S. Pat. No.5,534,615, expressly incorporated herein by reference, for example.

An “acidic variant” is a variant of a polypeptide of interest which ismore acidic (e.g. as determined by cation exchange chromatography) thanthe polypeptide of interest. An example of an acidic variant is adeamidated variant.

A “deamidated” variant of a polypeptide molecule is a polypeptidewherein one or more asparagine residue(s) of the original polypeptidehave been converted to aspartate, i.e. the neutral amide side chain hasbeen converted to a residue with an overall acidic character. DeamidatedhumMAb4D5 antibody from the Example below has Asn30 in CDR1 of either orboth of the V_(L) regions thereof converted to aspartate. The term“deamidated human DNase” as used herein means human DNase that isdeamidated at the asparagine residue that occurs at position 74 in theamino acid sequence of native mature human DNase (U.S. Pat. No.5,279,823; expressly incorporated herein by reference).

The term “mixture” as used herein in reference to a compositioncomprising an anti-HER2 antibody, means the presence of both the desiredanti-HER2 antibody and one or more acidic variants thereof. The acidicvariants may comprise predominantly deamidated anti-HER2 antibody, withminor amounts of other acidic variant(s). It has been found, forexample, that in preparations of anti-HER2 antibody obtained fromrecombinant expression, as much as about 25% of the anti-HER2 antibodyis deamidated.

In preferred embodiments of the invention, the polypeptide is arecombinant polypeptide. A “recombinant polypeptide” is one which hasbeen produced in a host cell which has been transformed or transfectedwith nucleic acid encoding the polypeptide, or produces the polypeptideas a result of homologous recombination.

“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing nucleic acid into a cell. Followingtransformation or transfection, the nucleic acid may integrate into thehost cell genome, or may exist as an extrachromosomal element. The “hostcell” includes a cell in in vitro cell culture as well a cell within ahost animal. Methods for recombinant production of polypeptides aredescribed in U.S. Pat. No. 15 5,534,615, expressly incorporated hereinby reference, for example.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The antibody herein is directed against an “antigen” of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those polypeptides discussed above.Preferred molecular targets for antibodies encompassed by the presentinvention include CD polypeptides such as CD3, CD4, CD8, CD19, CD20 andCD34; members of the HER receptor family such as the EGF receptor, HER2,HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1,p150,95, VLA-4, ICAM-1, VCAM and av/b3 integrin including either a or bsubunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies);growth factors such as VEGF; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; polypeptide Cetc. Soluble antigens or fragments thereof, optionally conjugated toother molecules, can be used as immunogens for generating antibodies.For transmembrane molecules, such as receptors, fragments of these (e.g.the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). In a further embodiment, “monoclonalantibodies” can be isolated from antibody phage libraries generatedusing the techniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques areviable alternatives to traditional monoclonal antibody hybridomatechniques for isolation of monoclonal antibodies. Alternatively, it isnow possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Polypeptides of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined. The CDR and FR residuesof the rhuMAb HER2 antibody of the example below (humAb4D5-8) areidentified in Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Varioustechniques have been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). In another embodiment, the F(ab′)₂ isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)₂molecule. According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner.

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185. “Single-chain Fv” or “sFv” antibodyfragments comprise the V_(H) and V_(L) domains of antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the sFv to form the desired structurefor antigen binding. For a review of sFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WC) 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Polypeptide Eng.8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

“Multispecific antibodies” have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two antigens(i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α(IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants; and BsAbs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase. Examples of trispecific antibodiesinclude anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques. Techniquesfor generating bispecific antibodies from antibody fragments have alsobeen described in the literature. For example, bispecific antibodies canbe prepared using chemical linkage. Brennan et al., Science, 229: 81(1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) diners has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

The phrase “ion exchange material” refers to a solid phase which isnegatively charged (i.e. a cation exchange resin) or positively charged(i.e. an anion exchange resin). The charge may be provided by attachingone or more charged ligands to the solid phase, e.g. by covalentlinking. Alternatively, or in addition, the charge may be an inherentproperty of the solid phase (e.g. as is the case for silica, which hasan overall negative charge).

By “solid phase” is meant a non-aqueous matrix to which one or morecharged ligands can adhere. The solid phase may be a purificationcolumn, a discontinuous phase of discrete particles, a membrane, orfilter etc. Examples of materials for forming the solid phase includepolysaccharides (such as agarose and cellulose); and other mechanicallystable matrices such as silica (e.g. controlled pore glass),poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles andderivatives of any of the above.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, BAKERBOND ABX™,sulphopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™or SP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from Pharmacia).

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., Ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 5 to about 7(e.g. as in Example 1 below). Examples of buffers that will control thepH in this range include MES, MOPS, MOPSO, phosphate, acetate, citrate,succinate, and ammonium buffers, as well as combinations of these.

The “loading buffer” is that which is used to load the compositioncomprising the polypeptide molecule of interest and one or morecontaminants onto the ion exchange resin. The loading buffer has aconductivity and/or pH such that the polypeptide molecule of interest(and generally one or more contaminants) is/are bound to the ionexchange resin.

The “intermediate buffer” is used to elute one or more contaminants fromthe ion exchange resin, prior to eluting the polypeptide molecule ofinterest. The conductivity and/or pH of the intermediate buffer is/aresuch that the contaminant is eluted from the ion exchange resin, but notsignificant amounts of the polypeptide of interest.

The term “wash buffer” when used herein refers to a buffer used to washor re-equilibrate the ion exchange resin, prior to eluting thepolypeptide molecule of interest. Conveniently, the wash buffer andloading buffer may be the same, but this is not required.

The “elution buffer” is used to elute the polypeptide of interest fromthe solid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all contaminants and thepolypeptide of interest from the ion exchange resin.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is mmhos (mS/cm),and can be measured using a conductivity meter sold, e.g., by Orion. Theconductivity of a solution may be altered by changing the concentrationof ions therein. For example, the concentration of a buffering agentand/or concentration of a salt (e.g. NaCl or KCl) in the solution may bealtered in order to achieve the desired conductivity. Preferably, thesalt concentration of the various buffers is modified to achieve thedesired conductivity as in the Example below.

By “purifying” a polypeptide from a composition comprising thepolypeptide and one or more contaminants is meant increasing the degreeof purity of the polypeptide in the composition by removing (completelyor partially) at least one contaminant from the composition. A“purification step” may be part of an overall purification processresulting in a “homogeneous” composition, which is used herein to referto a composition comprising at least about 70% by weight of thepolypeptide of interest, based on total weight of the composition,preferably at least about 80% by weight.

Unless indicated otherwise, the term “HER2” when used herein refers tohuman HER2 protein and “HER2” refers to human HER2 gene. The human HER2gene and HER2 protein are described in Semba et al., PNAS (USA)82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986)(Genebank accession number X03363), for example.

The term “humMAb4D5-8” when used herein refers to a humanized anti-HER2antibody comprising the light chain amino acid sequence of SEQ ID NO:1and the heavy chain amino acid sequence of SEQ ID NO:2 or amino acidsequence variants thereof which retain the ability to bind HER2 andinhibit growth of tumor cells which overexpress HER2 (see U.S. Pat. No.5,677,171; expressly incorporated herein by reference).

The “pI” or “isoelectric point” of a polypeptide refer to the pH atwhich the polypeptide's positive charge balances its negative charge. pIcan be calculated from the net charge of the amino acid residues of thepolypeptide or can be determined by isoelectric focussing (e.g. usingCSx chromatography as in the Example below).

By “binding” a molecule to an ion exchange material is meant exposingthe molecule to the ion exchange material under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the ion exchange material by virtue of ionic interactions between themolecule and a charged group or charged groups of the ion exchangematerial.

By “washing” the ion exchange material is meant passing an appropriatebuffer through or over the ion exchange material.

To “elute” a molecule (e.g. polypeptide or contaminant) from an ionexchange material is meant to remove the molecule therefrom by alteringthe ionic strength of the buffer surrounding the ion exchange materialsuch that the buffer competes with the molecule for the charged sites onthe ion exchange material.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with thepolypeptide purified as described herein. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel, toxotere, methotrexate, cisplatin,melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C,mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide,daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins,esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors, such astamoxifen and onapristone.

MODES FOR CARRYING OUT THE INVENTION

The invention herein provides a method for purifying a polypeptide froma composition (e.g. an aqueous solution) comprising the polypeptide andone or more contaminants. The composition is generally one resultingfrom the recombinant production of the polypeptide, but may be thatresulting from production of the polypeptide by peptide synthesis (orother synthetic means) or the polypeptide may be purified from a nativesource of the polypeptide. Preferably the polypeptide is an antibody,e.g. one which binds the HER2 antigen.

For recombinant production of the polypeptide, the nucleic acid encodingit is isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding thepolypeptide is readily isolated and sequenced using conventionalprocedures (e.g., where the polypeptide is an antibody by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence (e.g. as described in U.S. Pat.No. 5,534,615, specifically incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated polypeptide arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for polypeptide production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the polypeptide of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells.

In addition, any of the media described in Ham et al., Meth. Enz. 58:44(1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430;WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture mediafor the host cells. Any of these media may be supplemented as necessarywith hormones and/or other growth factors (such as insulin, transferrin,or epidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

When using recombinant techniques, the polypeptide can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the polypeptide is produced intracellularly, as a first step,the particulate debris, either host cells or lysed cells (e.g. resultingfrom homogenization), is removed, for example, by centrifugation orultrafiltration. Where the polypeptide is secreted into the medium,supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit.

The polypeptide is then subjected to one or more purification steps,including the ion exchange chromatography method as claimed herein.Examples of additional purification procedures which may be performedprior to, during, or following the ion exchange chromatography methodinclude fractionation on a hydrophobic interaction chromatography (e.g.on phenyl sepharose), ethanol precipitation, isoelectric focusing,Reverse Phase HPLC, chromatography on silica, chromatography on HEPARINSEPHAROSE™, further anion exchange chromatography and/or further cationexchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfateprecipitation, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography (e.g. using protein A, protein G,an antibody, a specific substrate, ligand or antigen as the capturereagent).

Ion exchange chromatography is performed as claimed herein. A decisionis first made as to whether an anion or cation exchange resin is to beemployed. In general, a cation exchange resin may be used forpolypeptides with pIs greater than about 7 and an anion exchange resinmay be used for polypeptides with pIs less than about 7.

The anion or cation exchange resin is prepared according to knownmethods. Usually, an equilibration buffer is passed through the ionexchange resin prior to loading the composition comprising thepolypeptide and one or more contaminants onto the resin. Conveniently,the equilibration buffer is the same as the loading buffer, but this isnot required.

The various buffers used for the chromatography depend, for example, onwhether a cation or anion exchange resin is employed. This is shown moreclearly in the flow diagrams of FIGS. 1 and 2.

With particular reference to FIG. 1, which shows exemplary steps to beperformed where a cation exchange resin is used, the pH and/orconductivity of each buffer is/are increased relative to the precedingbuffer, except for the wash buffer where the conductivity and/or pHis/are less than the conductivity and/or pH of the precedingintermediate buffer. The aqueous solution comprising the polypeptide ofinterest and contaminant(s) is loaded onto the cation exchange resinusing the loading buffer that is at a pH and/or conductivity such thatthe polypeptide and the contaminant bind to the cation exchange resin.As in the Example below, the loading buffer may be at a first lowconductivity (e.g. from about 5.2 to about 6.6 mmhos). An exemplary pHfor the loading buffer may be about 5.0 (see FIG. 1). From about 20mg/mL to about 35 mg/mL of the polypeptide (e.g. of a full lengthantibody) may, for example, be loaded on the ion exchange resin.

The cation exchange resin is then washed with an intermediate bufferwhich is at a second conductivity and/or pH so as to essentially elutethe contaminant, but not a substantial amount of the polypeptide ofinterest. This may be achieved by increasing the conductivity or pH, orboth, of the intermediate buffer. The change from loading buffer tointermediate buffer may be step-wise or gradual as desired. In theExample herein, the intermediate buffer had a greater conductivity thanthat of the loading buffer (i.e. the intermediate buffer's conductivitywas in the range from about 7.3 to about 8.4 mmhos). Alternatively, asshown in FIG. 1, the pH of the intermediate buffer may exceed that ofthe loading buffer in this embodiment of the invention, where a cationexchange resin is used. For example, the intermediate buffer may have apH of about 5.4.

Following washing with the intermediate buffer, the cation exchangeresin is washed or re-equilibrated with the wash buffer which has aconductivity or pH, or both, which is/are less than that of theintermediate buffer (i.e. the conductivity, or pH, or both, is/arechanged in an opposite, i.e. reverse, direction to the preceding step,unlike ion exchange chromatography steps in the literature). In theExample below, the wash buffer had about the same conductivity as theloading buffer (i.e. in the range from about 5.2 to about 6.6 mmhos) andits conductivity was, therefore, less than that of the intermediatebuffer. In another embodiment, one may reduce the conductivity of thewash buffer to a conductivity that is less than, or greater than, thatof the loading buffer, provided the conductivity of the wash buffer isless than that of the intermediate buffer. In another embodiment, the pHof the wash buffer may be less than the pH of the intermediate buffer(e.g. the pH of the wash buffer may about 5.0). The change inconductivity and/or pH of the wash buffer compared to the intermediatebuffer may be achieved by step-wise or gradual change of either or bothof these parameters.

After the wash step of the preceding paragraph, the cation exchangeresin is prepared for elution of the desired polypeptide moleculetherefrom. This is achieved using an elution buffer that has a pH and/orconductivity such that the desired polypeptide no longer binds to thecation exchange resin and therefore is eluted therefrom. The pH and/orconductivity of the elution buffer generally exceed(s) the pH and/orconductivity of the loading buffer, the intermediate buffer and the washbuffer used in the previous steps. In the Example below, theconductivity of the elution buffer was in the range from about 10.0 toabout 11.0 mmhos. Alternatively, or in addition, the pH of the elutionbuffer may be increased relative to the wash buffer and to theintermediate buffer (for example, the pH of the elution buffer may about6.0). The change in conductivity and/or pH may be step-wise or gradual,as desired. Hence, the desired polypeptide is retrieved from the cationexchange resin at this stage in the method.

In an alternative embodiment, the ion exchange material comprises ananion exchange resin. This embodiment of the invention is depicted inFIG. 2 herein. As illustrated in this figure, the changes inconductivity are generally as described above with respect to a cationexchange resin. However, the direction of change in pH is different foran anion exchange resin. For example, if elution of the contaminant(s)and polypeptide are to be achieved by altering pH, the loading bufferhas a first pH and the pH is decreased in the intermediate buffer so asto elute the contaminant or contaminants. In the third step, the columnis washed/re-equilibrated with the wash buffer and the change inconductivity or pH, or both, is in the opposite direction to that of theprevious step. Hence, the pH may be increased in the wash buffer,compared to the intermediate buffer. Following this step, thepolypeptide of interest is eluted from the anion exchange resin using anelution buffer at a fourth conductivity and/or pH. If pH is altered, itwill normally be less than the pH of the loading buffer, theintermediate buffer and the wash buffer. The change in pH and/orconductivity in progressive buffers can, as explained above, bestep-wise or gradual.

In the preferred embodiment of the invention, a single parameter (i. e.either conductivity or pH) is changed to achieve elution of both thepolypeptide and contaminant, while the other parameter (i.e. pH orconductivity, respectively) remains about constant. For example, whilethe conductivity of the various buffers (loading buffer, intermediatebuffer, wash buffer and/or elution buffer) may differ, the pH's thereofmay be essentially the same.

In an optional embodiment of the invention, the ion exchange resin isregenerated with a regeneration buffer after elution of the polypeptide,such that the column can be re-used. Generally, the conductivity and/orpH of the regeneration buffer is/are such that substantially allcontaminants and the polypeptide of interest are eluted from the ionexchange resin. Generally, the regeneration buffer has a very highconductivity for eluting contaminants and polypeptide from the ionexchange resin.

The method herein is particularly useful for resolving a polypeptidemolecule of interest from at least one contaminant, where thecontaminant and polypeptide molecule of interest differ only slightly inionic charge. For example, the pIs of the polypeptide and contaminantmay be only “slightly different”, for example they may differ by onlyabout 0.05 to about 0.2 pI units. In the Example below, this methodcould be used to resolve an anti-HER2 antibody having a pI of 8.87, froma singly-deamidated variant thereof having a pI of 8.79. Alternatively,the method may be used to resolve a deamidated DNase, for example, fromnondeamidated DNase. In another embodiment, the method may be used toresolve a polypeptide from a glycosylation variant thereof, e.g. forresolving a variant of a polypeptide having a different distribution ofsialic acid compared to the nonvariant polypeptide.

The polypeptide preparation obtained according to the ion exchangechromatography method herein may be subjected to additional purificationsteps, if necessary. Exemplary further purification steps have beendiscussed above.

Optionally, the polypeptide is conjugated to one or more heterologousmolecules as desired. The heterologous molecule may, for example, be onewhich increases the serum half-life of the polypeptide (e.g.polyethylene glycol, PEG), or it may be a label (e.g. an enzyme,fluorescent label and/or radionuclide) or a cytotoxic molecule (e.g. atoxin, chemotherapeutic drug, or radioactive isotope etc).

A therapeutic formulation comprising the polypeptide, optionallyconjugated with a heterologous molecule, may be prepared by mixing thepolypeptide having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.“Pharmaceutically acceptable” carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) ThehumMAb4D5-8 antibody of particular interest herein may be prepared as alyophilized formulation, e.g. as described in WO 97/04801; expresslyincorporated herein by reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended. For example, for an anti-HER2antibody a chemotherapeutic agent, such as a taxoid or tamoxifen, may beadded to the formulation.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulation to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide variant, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The polypeptide purified as disclosed herein or the compositioncomprising the polypeptide and a pharmaceutically acceptable carrier isthen used for various diagnostic, therapeutic or other uses known forsuch polypeptides and compositions. For example, the polypeptide may beused to treat a disorder in a mammal by administering a therapeuticallyeffective amount of the polypeptide to the mammal.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLE 1

Full length human IgG rhuMAb HER2 (humAb4D5-8 in Carter et al. Proc.Natl. Acad. Sci. 89: 4285-4289 (1992) comprising the light chain aminoacid sequence of SEQ ID NO:l and heavy chain amino acid sequence of SEQID NO:2) was produced recombinantly in CHO cells. Following proteinproduction and secretion to the cell culture medium, the CHO cells wereseparated from the cell culture medium by tangential flow filtration(PROSTACK™). Protein A chromatography was then performed by applying theHarvested Cell Culture Fluid (HCCF) from the CHO cells directly to anequilibrated PROSEP A™ column (Bioprocessing, Ltd).

Following Protein A chromatography, cation exchange chromatography wasperformed using a sulphopropyl (SP)-SEPHAROSE FAST FLOW™ (SPSFF) column(Pharmacia) to further separate the desired anti-HER2 antibody molecule.The chromatography operation was performed in bind and elute mode.

The SPSFF column was prepared for load by sequential washes withregeneration buffer (0.025 M MES/1.0 M NaCl, pH 5.6) followed byequilibration buffer (0.025 M MES/50 mM NaCl, pH 5.6). The column wasthen loaded with Protein A pool adjusted to a pH of 5.60±0.05 and aconductivity of 5.8±0.2 mmhos. Prior to elution, the column was washedin three steps: (1) loading buffer (0.025 M MES/50 mM NaCl, pH 5.6) fora minimum of 1 column volume; (2) intermediate buffer (0.025 M MES/70 mMNaCl, pH 5.6) until an apex of a 280 nm peak was reached; and (3) washbuffer (0.025 M MES/50 mM NaCl, pH 5.6) for a minimum of 1.2 columnvolumes. rhuMAb HER2 was then eluted from the column with elution buffer(0.025 M MES/95 mM NaCl, pH 5.6). The elution 280 nm profile has ashoulder on the leading edge (FIG. 3). At the inflection point of thisshoulder, pooling starts and continues for an additional 5 columnvolumes. The column was then regenerated with regeneration buffer (0.025M MES/1.0 M NaCl, pH 5.6).

Materials and Methods

Column and Load Preparation:

A reduced-scale SPSFF column was packed. The dimensions were: 27.0 mLvolume, 1.0 cm diameter and 34.5 cm bed height. The pH of an aliquot ofProtein A pool was titered to 5.6 with 1.5 M Tris base. The conductivityof the pool was reduced by the addition of an equal volume of sterilewater for injection (SWFI).

Chromatography:

The chromatography runs for this study were performed with Pharmacia'sUNICORN™ FPLC system. The equilibration, load, and initial wash stepswere performed at a linear flow rate of 200 cm/h. All chromatographysteps were performed at a linear flow rate of 100 cm/h. The sequence ofchromatography steps are defined in Table 1. A total of sixchromatography runs were performed with load densities of 15, 20, 25,30, 35, and 40 mg of rhuMAb HER2 per mL of SPSFF resin.

TABLE 1 Chromatography Steps¹ Chromatography Approximate Step BufferEndpoint Equilibration: 0.025M MES/1.0M NaCl, 2 CV² Part 1 pH 5.6Equilibration: 0.025M MES/0.05M NaCl, pH: 5.6 ± 0.1 Part 2 pH 5.6 Cond.:5.8 ± 0.2 mmhos Load Adjusted Protein A Pool As Required Wash 1 0.025MMES/0.05M NaCl, 1.5 CV pH 5.6 Wash 2 0.025M MES/0.07M NaCl, Apex of PeakpH 5.6 Wash 3 0.025M MES/0.05M NaCl, 2 CV pH 5.6 Elution: Prepool 0.025MMES/0.095M NaCl, To Leading Shoulder's pH 5.6 Inflection Point (˜1.2 CV)Elution: Pool 0.025M MES/0.095M NaCl, 5 CV pH 5.6 Regeneration 0.025MMES/1.0M NaCl, 2 CV pH 5.6 ¹The equilibration of the resin was performedin manual mode; the remaining steps were executed from a PharmaciaUnicom Program. ²CV = column volume(s).

Total Protein:

The protein concentration of each chromatography fraction (flow through,wash steps, elution prepool, elution pool, and regeneration) wasdetermined by spectrophometric scans of each sample. The results wereused to calculate product recovery yields. The extinction coefficientfor rhuMAb HER2 is 1.45. Calculations used to derive the results (FIG.4) are:${{Protein}\quad {Concentration}\quad \text{(}{mg}\text{/}{mL}\text{)}} = {\frac{280\quad {nm}}{1.45} \times {Dilution}\quad {Factor}}$

Protein Mass (mg) in Each Fraction=Protein Concentration(mg/mL)×Fraction Volume (mL)${{Yield}\quad (\%)} = {\frac{{Fraction}\quad {Mass}\quad ({mg})}{{Total}\quad {Mass}\quad ({mg})} \times 100}$

Determination of rhuMAb HER2 Antibody Variants (CSx HPIEX):

The rhuMAb HER2 SPSFF chromatography column resolves antibody variants.Fractions from each of the study chromatographies were tested for therelative amount of variant antibody by CSx HPIEX chromatography. ABAKERBOND WIDE-PORET™ CSx HPIEX column (4.6×250 mm) was run at 1 mL/minat 55° C. The mobile phase was formed from a tertiary gradient (Table2).

TABLE 2 Gradient Scheme Time (min) % A % B % C 0-Initial Conditions 49 150 10.0 40 10 50 50.0 33 17 50 50.2 49 1 50 70.0 49 1 50 The column isrun at 1 ml/min at 55° C.

The A buffer was 0.025 M MES, pH 5.9; the B buffer was 1 M AmmoniumAcetate, pH 7.0; and the C solution was sterile water for injection. Thecolumn was equilibrated with the gradient's initial conditions (49% A;1% B; and 50% C) and 200 μl of sample, diluted. with SWFI and containing<300 μg protein, was injected. Each resulting chromatogram wasintegrated to determine the percent area of each peak for each fraction(Table 3 and FIG. 5).

TABLE 3 CSx HPIEX analysis of rhuMAb HER2 CSx Peak rhuMAb HER2 Variant a& b Light Chain: Asn→Asp³⁰ deamidation -and- Other unidentifiablevariation by tryptic map 1 Light Chain: Asn→Asp³⁰ deamidation 3 FullyProcessed Antibody 4 Heavy Chain: Asp→Iso-Asp¹⁰² -and/or- Heavy Chain:An Additional Lys⁴⁵⁰ Others Heavy Chain: Asp→Succinimide¹⁰² -and/or-Multiple permutations found in Peaks 1 and 4

Chromatograms Compared:

The absorbance data (AU 280 nm) from each chromatography file wasexported from Unicorn in ASCII format. The data from the 0.025 MMES/0.07 M NaCl, pH 5.6 wash was translated into Excel format and copiedinto KALEIDAGRAPH™. Using KALEIDAGRAPH™, the wash profiles were overlaid(FIG. 6) and compared to each other.

RESULTS AND DISCUSSION

Deamidated and other acidic variants of rhuMAb HER2 were produced whenthe antibody was made by recombinant DNA technology (see e.g., CSx peaksa, b and 1 in FIG. 5). The deamidated and other acidic variantsconstituted about 25% (calculated as area under the integrated curve orprofile obtained by CSx chromatography) of the composition obtained fromthe initial Protein A chromatography step. It was discovered that theion exchange method described herein could be used to substantiallyreduce the amount of deamidated and other acidic variants in theanti-HER2 composition, i.e. to about 13% or less (i.e. the amount ofacidic variants in the preparation subjected to cation exchangechromatography as described herein was decreased by about 50% or more).

An absorbance trace from a cation exchange column run performed asdescribed above is shown in FIG. 3. This method resolved a deamidatedvariant of anti-HER2 antibody that differed only slightly fromnondeamidated anti-HER2 antibody. The increase in conductivity from theinitial conditions to the intermediate wash began to elute thedeamidated anti-HER2 antibody. However, continued washing at thisconductivity was found to elute nondeamidated anti-HER2 antibody,resulting in a loss of product. Proceeding directly from theintermediate buffer to the elution buffer was observed to result ineither an unacceptably low removal of deamidated anti-HER2 antibody fromthe product if pooling began early or unacceptably low yields ofanti-HER2 antibody product if pooling was delayed until the deamidatedanti-HER2 antibody was reduced. It was discovered that by going back tolower conductivity as used initially, the elution of deamidatedanti-HER2 antibody continued, without significant anti-HER2 antibodyproduct elution.

The effect of rhuMAb HER2 load on (a) buffer requirements, (b) productrecovery in the pool, and (c) product quality in the pool was evaluated.

At load densities of 15 mg/mL up to 35 mg/mL, the product yield in theelution pool is approximately 75%. For the load density of 40 mg/mL, theproduct yield in the pool dropped to 65% (FIG. 4). This reduced recoveryin the pool is largely attributed to an increased antibody in the twowash steps (at 70 mM NaCl and 50 mM NaCl, respectively).

The quality of rhuMAb HER2 in all the elution pools is equivalent asdetermined by CSx HPIEX analysis (FIG. 5). Compared to the loadmaterial; there is an enrichment of the nondeamidated antibody (Peak 3),no change in the amount Iso-Asp¹⁰² or Lys⁴⁵⁰ antibody (Peak 4), and areduction of the amount of Asp³⁰ deamidated antibody (Peaks a, b, 1 andothers).

The quality of rhuMAb HER2 in these cation pools is improved through theintermediate wash step. As the mass of rhuMAb HER2 bound to the resinincreases, the intermediate buffer volume consumption needed to reachthe apex of the 280 nm peak decreases. The buffer volume required for a40 mg/mL load density is approximately 2.5 column volumes. The buffervolume required for a 15 mg/mL load density is approximately 15 columnvolumes. The exact increase of buffer requirement is not linear with the5 mg/mL incremental changes between these two extremes. The greatestincrease is seen between the load densities of 20 mg/mL and 15 mg/mL.Here the requirement doubles from 7.5 column volumes to the previouslymentioned 15 column volumes of buffer. If the apex of the 70 mM NaClwash peak is reached, however, the product quality is equivalent for anyof load densities examined.

This study determined how much rhuMAb HER2 can be loaded onto the SPSFFresin. Between the ranges of 15 to 40 mg of antibody per mL of resin,there is no difference in the quality of rhuMAb HER2 recovered in theelution pool. The quantity of rhuMAb HER2 recovered, however, is reducedby approximately 10% when the resin is loaded with greater than 35mg/mL. For consistent yields it is recommended that 35 mg/mL be set asthe maximum load for manufacture of rhuMAb HER2. Furthermore, due to thesubstantial increase in the 70 mM NaCl wash volume requirement betweenthe 20 and 15 mg/mL; it is recommended that 20 mg/mL be set as theminimal load for manufacture of rhuMAb HER2.

2 1 214 PRT Artificial sequence Sequence is synthesized. 1 Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp ArgVal Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 20 25 30 Thr Ala Val AlaTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr SerAla Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser ArgSer Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu AspPhe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 His Tyr Thr Thr Pro Pro Thr PheGly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys Arg Thr Val Ala Ala ProSer Val Phe Ile Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser GlyThr Ala Ser Val Val Cys Leu 125 130 135 Leu Asn Asn Phe Tyr Pro Arg GluAla Lys Val Gln Trp Lys Val 140 145 150 Asp Asn Ala Leu Gln Ser Gly AsnSer Gln Glu Ser Val Thr Glu 155 160 165 Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser Thr Leu Thr 170 175 180 Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala Cys Glu 185 190 195 Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe Asn 200 205 210 Arg Gly Glu Cys 2 449 PRTArtificial sequence Sequence is synthesized. 2 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30 Asp Thr Tyr Ile His Trp ValArg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Arg Ile Tyr ProThr Asn Gly Tyr Thr Arg Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe ThrIle Ser Ala Asp Thr Ser 65 70 75 Lys Asn Thr Ala Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly GlyAsp Gly Phe Tyr 95 100 105 Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ser 110 115 120 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Ser Ser 125 130 135 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys Leu Val Lys 140 145 150 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpAsn Ser Gly Ala 155 160 165 Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser Ser 170 175 180 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 185 190 195 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser 200 205 210 Asn Thr Lys Val Asp Lys Lys Val Glu Pro LysSer Cys Asp Lys 215 220 225 Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro LysAsp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp TyrVal Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp 305 310 315 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala 320 325 330 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln 335 340 345 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Glu Glu 350 355 360 Met Thr Lys Asn Gln Val Ser Leu Thr Cys LeuVal Lys Gly Phe 365 370 375 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly Gln Pro 380 385 390 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp Ser Asp Gly 395 400 405 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp 410 415 420 Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu 425 430 435 His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly 440 445

What is claimed is:
 1. A composition comprising a mixture of anti-HER2antibody and one or more acidic variants thereof, wherein the amount ofthe acidic variant(s) is less than about 25%.
 2. The composition ofclaim 1 further comprising a pharmaceutically acceptable carrier.
 3. Thecomposition of claim 1 wherein the anti-HER2 antibody is humMAb4D5-8.